In Situ Metabolic Rates of Alkane-Degrading Sulphate-Reducing Bacteria in Hydrocarbon Seep Sediments Revealed by Combining CARD-FISH, NanoSIMS, and Mathematical Modelling

Abstract

Marine hydrocarbon seeps are hotspots for sulphate reduction coupled to hydrocarbon oxidation. In situ metabolic rates of sulphate-reducing bacteria (SRB) degrading hydrocarbons other than methane, however, remain poorly understood. Here, we assessed the environmental role of Desulfosarcinaceae clades SCA1, SCA2 for degradation of n-butane and clade LCA2 for n-dodecane. Quantification by CARD-FISH showed that SCA1 constituted up to 31%, SCA2 up to 9%, and LCA2 up to 6% of cells from the recently re-classified class Deltaproteobacteria across diverse hydrocarbon seeps. Cell-specific oxidation rates estimated by stable-isotope probing combined with NanoSIMS and modelling were ~0.73 and ~2.11 fmol butane cell^-1 d^-1 for SCA1 and SCA2, respectively, and ~0.023 fmol dodecane cell^-1 d^-1 for LCA2 in sediments from Amon Mud Volcano and Guaymas Basin sediments. Cellular carbon assimilation, dissolved inorganic carbon production, and sulphate reduction rates indicated that butane-degrading SRB have higher metabolic activity than those utilising dodecane. Estimates based on in situ cell abundances, biovolumes, and cellular activities suggest that at certain seeps, clades SCA1, SCA2 and LCA2 account for nearly all sulphate reduction not driven by methane oxidation. These findings highlight the important role of alkane-degrading SRB in influencing marine carbon and sulphur cycles, particularly at seeps emitting higher hydrocarbons.

Keywords: NanoSIMS; activity; alkane degraders; anaerobic butane degradation; anaerobic dodecane degradation; cell numbers; hydrocarbon seeps; marine sediments; stable‐isotope probing.

FIGURE 1

Characteristics of the relevant carbon pools in sediment slurries incubated with added ¹³C‐alkane (butane or dodecane). The shown characteristics include (A) porewater alkane concentrations, (B) ¹³C enrichment of the porewater DIC, (C) ¹³C enrichment (left axis, filled symbols) and concentration (right axis, open symbols) of the bulk TOC, (D) cell counts of the target cells, and (E) ¹³C enrichment of the target cells (data point is the mean value, error‐bar corresponds to 1 × SD of n individual cells). The ¹³C enrichment data are presented as excess ¹³C atom fractions in atom %. In all panels, points show experimental data while lines show modelled data. The modelled data assumed that the biomass of the alkane degrading population was the same as derived from the measured abundance and biovolume of the target cells (dashed line), or was increased by a factor BIF (solid line). Alkane concentrations (A), DIC ¹³C enrichment (B) and cell abundance data (D) were taken from Kleindienst et al. (2014).

FIGURE 2

Correlative imaging of target cells and their alkane‐derived carbon assimilation activity. (A) CARD‐FISH images. (B) Corresponding NanoSIMS images of ¹³C atom fraction. (C) Summary of excess ¹³C atom fractions in all target cells measured by NanoSIMS in this study. Shown are data for specific butane‐ or dodecane‐degrading cells from Amon MV and Guaymas Basin seep sediments identified by specific probes for SCA1, SCA2, and LCA2. In panel B, note differences in colour scales among the sites. Scale bars represent 2 μm.

FIGURE 3

NanoSIMS images of selected aggregate‐forming butane‐degrading target cells (group SCA1) from Amon Mud Volcano marine seep sediments. Shown are ¹³C atom fraction images of cells incubated with ¹³C‐butane for 9 days (A, B) and 15 days (C, D). The apparent enrichment in ¹³C was similar among cells within one aggregate, whereas it differed between aggregates, suggesting at least two subpopulations with distinct levels of substrate assimilation. Scale bar indicates 2 μm.